Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing

PURPOSE: To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma. SETTING: Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom. METHODS: Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts. RESULTS: The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF. CONCLUSIONS: Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.     

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.     

In vitro effects of three blood derivatives on human corneal epithelial cells

Purpose. We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. Methods. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. Results. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. Conclusions. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Lacrimal gland HGF, KGF, and EGF mRNA levels increase after corneal epithelial wounding.

PURPOSE: To evaluate the effect of corneal epithelial wounding on lacrimal gland expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and epidermal growth factor (EGF) in the rabbit model. METHODS: Rabbits had corneal epithelial scrape injuries, and the lacrimal gland was removed at different times after wounding. HGF, KGF, and EGF mRNA expression was examined by quantitative RNase protection assay. HGF, KGF, and EGF proteins were detected in rabbit lacrimal tissue using immunoprecipitation and western blot analysis. RESULTS: HGF mRNA and EGF mRNA were significantly increased in rabbit lacrimal gland tissue within 8 hours after corneal epithelial injury. The increase in KGF mRNA expression was small and reached significance I clay after corneal injury. Lacrimal gland expression peaked at 3 days after wounding for each growth factor mRNA, the same day, on average, that the epithelial defect healed. After the peak increase in expression, there was a progressive decline in expression of each growth factor mRNA, but production was still increased compared with prewound levels. HGF protein, KGF protein, and EGF proteins were detected in rabbit lacrimal gland tissue. CONCLUSIONS: Levels of HGF, KGF, and EGF mRNAs increase in rabbit lacrimal gland tissue in response to corneal epithelial wounding. The results of this study are consistent with the existence of a cornea-nervous system-lacrimal gland regulatory loop modulating expression of these growth factor mRNAs. The lacrimal gland is a likely source of increased HGF and EGF proteins detected in tears in previous studies.     

Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing

EGF is an essential growth factor needed for epithelial cell proliferation and wound healing of the cornea, but the molecular mechanism is not understood. Although studies have shown that EGF in some non-phagocytic cells induces ROS generation, little is known about the role of ROS in corneal epithelial cells. Therefore, we examined the potential physiological role of ROS in corneal cell proliferation, adhesion and wound healing using rabbit or human corneal epithelial cells, and pig whole cornea organ culture as models. EGF (5 ng/ml)-induced ROS in serum-starved RCE or HCE cells were captured as DCFH fluorescence and detected by confocal microscopy. The elevation of ROS was eradicated when the cells were pretreated with an antioxidant N-acetylcysteine (NAC) or mannitol, or with inhibitor to NADPH oxidase (DPI), or to lipoxygenase (NDGA). EGF-induced ROS generation correlated with cell growth and activation of Akt and MAPK signaling pathways, while NAC eliminated all these effects. EGF-stimulated cell adhesion or migration in cell culture was greatly suppressed in the presence of NAC while EGF-facilitated epithelial cell wound healing in corneal organ culture was also blocked by NAC. This is the first demonstration of a novel ROS physiological function in corneal wound healing.     

血小板活化因子致角膜上皮伤口迟愈合的实验研究

目的　利用兔去上皮角膜模型 ,研究血小板活化因子 (PAF)对角膜伤口愈合的作用及其分子生物学机制。方法　离体角膜上做正中直径 7mm圆形去上皮角膜伤口。去上皮角膜分为 3组 ,即对照、PAF及BN (PAF拮抗剂 )组 ,培养 4 8h后 ,行角膜上皮染色观察伤口愈合状况。分别对兔角膜上皮 (RCE)和角膜基质 (RCK)细胞进行体外传代培养 ,RCE和RCK细胞经PAF和 (或 )BN处理 ,培养 2 4h ,提纯RNA。应用RT PCR及核酸杂交技术分别检测肝细胞生长因子 (HGF)、角质形成生长因子 (KGF)及表皮生长因子 (EGF)基因在RCK和RCE细胞及HGF受体 (HGF R)基因在RCE细胞中的表达强度。分别应用CyQUANT荧光结合和Boyden小房技术检测PAF对RCE细胞黏附、增殖和迁徙的影响。结果　PAF (10 0nmol/L )明显抑制角膜上皮伤口愈合 ,4 8h对照、PAF和BN组角膜上皮未愈合面积经电脑图像分析分别为 :(6 0± 1.5 )U、(5 8 0± 7 0 )U和 (5 0± 1 0 )U。PAF明显增强RCE细胞黏附作用 ,对照、PAF和BN组每 96孔板贴附细胞数荧光光度平均值分别为 :36 96± 372、790 8± 6 71和 3487± 32 4。RT PCR结果显示 :PAF使HGFmRNA在RCK的表达强度降低 4 .1倍 ,同时明显减弱HGF R在RCE细胞中的表达 ,核酸杂交实验证实PCR结果。结论PAF明显增强RCE细胞的黏附作用 ,     

角质细胞生长因子促进角膜上皮损伤修复的研究

目的寻找促进角膜上皮损伤修复，治疗持续性角膜上皮缺损的有效方法。方法用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入及液体闪烁技术，观察不同浓度的外源性角质细胞生长因子（ｋｅｒａｔｉｎｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ，ＫＧＦ）对体外培养的人角膜上皮细胞生长的影响，由此推算出有效滴眼液浓度并应用于兔眼。用计算机图形分析系统计算角膜上皮愈合速率；用光镜和电镜评估愈合的质量。结果０．１～１００ｎｇ／ｍｌＫＧＦ有明显促进体外培养的人角膜上皮细胞生长的作用（增长率为２７．６６％～７６．７３％），且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１μｇ／ｍｌＫＧＦ滴兔眼，加速了角膜上皮损伤修复（愈合速率，ＫＧＦ组为１．７７±０．２３ｍｍ２／ｈ，与对照组１．４９±０．２４ｍｍ２／ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促生长作用，其滴眼液有加速兔眼角膜上皮创伤修复的作用。     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by keratinocyte growth factor

PURPOSE: The possible detrimental effect of hypoxia on the barrier function of corneal epithelial cells and whether keratinocyte growth factor (KGF) might protect against such an effect were investigated. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured for 4 days to allow the establishment of barrier function. They were then deprived of serum for 24 hours before exposure to 1% (hypoxia) or 21% (normoxia) oxygen for 24 hours. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). The localization of ZO-1 and occludin was determined by immunofluorescence microscopy, and the expression of these tight junctional proteins as well as the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK were examined by immunoblot analysis. RESULTS: Hypoxia induced a decrease in the TER of HCE cells compared with that of cells maintained under normoxia. The localization of ZO-1 at cell-cell borders was disrupted by hypoxia, whereas the distribution of occludin was not affected. Hypoxia also induced the downregulation of ZO-1 and a decrease in the phosphorylation of ERK without affecting the phosphorylation of p38 or JNK. All these effects of hypoxia were inhibited by KGF. The effects of KGF on TER and ZO-1 localization in cells exposed to hypoxia were inhibited by PD98059, an inhibitor of ERK signaling. Neither hypoxia nor KGF exhibited mitogenic or cytotoxic effects in HCE cells. CONCLUSIONS: Hypoxia induces disruption of the barrier function of HCE cells by eliciting the redistribution and degradation of ZO-1, and this effect is inhibited by KGF in a manner dependent on ERK activation.     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by hepatocyte growth factor

The barrier function of the corneal epithelium maintains corneal homeostasis and is mediated by tight junctions (TJs) and adherens junctions (AJs). It is also susceptible to disruption by hypoxia. We have now examined the effects of hypoxia on TJs and AJs as well as on barrier function in human corneal epithelial (HCE) cells. Moreover, we investigated whether such effects of hypoxia might be modulated by hepatocyte growth factor (HGF). The subcellular distribution of the TJ proteins ZO-1 and occludin, the AJ proteins E-cadherin and β-catenin, and actin filaments was examined by fluorescence microscopy. The abundance of junctional proteins as well as of myosin light chain kinase (MLCK) was determined by immunoblot analysis. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). Hypoxia-induced both the disappearance of ZO-1 from the borders of neighboring HCE cells as well as the down-regulation of ZO-1 expression without affecting the distribution or abundance of occludin, E-cadherin, or β-catenin. It also induced the formation of actin stress fibers, the up-regulation of MLCK expression, and a reduction in the TER of HCE cells. All these effects of hypoxia were inhibited by HGF. Neither hypoxia nor HGF exhibited a mitogenic or cytotoxic effect on HCE cells. HGF thus protects HCE cells from hypoxia-induced disruption of barrier function by maintaining the expression and distribution of ZO-1. Inhibition of the effects of hypoxia on the organization of the actin cytoskeleton might also contribute to this protective action of HGF.     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

Growth factor mRNA and protein in preserved human amniotic membrane

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.     

Growth factors: importance in wound healing and maintenance of transparency of the cornea

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF,and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.